Histone deacetylase inhibitors based on alpha-ketoepoxide compounds

ABSTRACT

Histone deacetylase is a metallo-enzyme with zinc at the active site. Compounds having a zinc-binding moiety, for example, an alpha-ketoepoxide group, such as an alpha-ketothio group, can inhibit histone deacetylase. Histone deacetylase inhibition can repress gene expression, including expression of genes related to tumor suppression. Accordingly, inhibition of histone deacetylase can provide an alternate route for treating cancer, hematological disorders, e.g., hemoglobinopathies, autosomal dominant disorders, e.g. spinal muscular atrophy and Huntington&#39;s disease, genetic related metabolic disorders, e.g., cystic fibrosis and adrenoleukodystrophy, or to stimulate hematopoietic cells ex vivo.

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No.10/442,177, filed on May 21, 2003, allowed, which claims priority under35 USC §119(e) to U.S. Patent Application Ser. No. 60/382,089, filed onMay 22, 2002, the entire contents of which is hereby incorporated byreference.

TECHNICAL FIELD

This invention relates to alpha-ketoepoxide compounds, and moreparticularly to alpha-ketoepoxide compounds that are histone deacetylaseinhibitors.

BACKGROUND

DNA in the nucleus of the cell exists as a hierarchy of compactedchromatin structures. The basic repeating unit in chromatin is thenucleosome. The nucleosome consists of a histone octamer of proteins inthe nucleus of the cell around which DNA is wrapped twice. The orderlypackaging of DNA in the nucleus plays an important role in thefunctional aspects of gene regulation. Covalent modifications of thehistones have a key role in altering chromatin higher order structureand function and ultimately gene expression. The covalent modificationof histones, such as acetylation, occurs by enzymatically mediatedprocesses.

Regulation of gene expression through the inhibition of the nuclearenzyme histone deacetylase (HDAC) is one of several possible regulatorymechanisms whereby chromatin activity can be affected. The dynamichomeostasis of the nuclear acetylation of histones can be regulated bythe opposing activity of the enzymes histone acetyl transferase (HAT)and histone deacetylase (HDAC). Transcriptionally silent chromatin canbe characterized by nucleosomes with low levels of acetylated histones.Acetylation reduces the positive charge of histones, thereby expandingthe structure of the nucleosome and facilitating the interaction oftranscription factors with the DNA. Removal of the acetyl group restoresthe positive charge, condensing the structure of the nucleosome. Histoneacetylation can activate DNA transcription, enhancing gene expression.Histone deacetylase can reverse the process and can serve to repressgene expression. See, for example, Grunstein, Nature 389, 349-352(1997); Pazin et al., Cell 89, 325-328 (1997); Wade et al., TrendsBiochem. Sci. 22, 128-132 (1997); and Wolffe, Science 272, 371-372(1996).

SUMMARY

Histone deacetylase is a metallo-enzyme with zinc at the active site.Compounds having a zinc-binding moiety, for example, analpha-ketoepoxide group, can inhibit histone deacetylase. Histonedeacetylase inhibition can alter gene expression, including expressionof genes related to tumor suppression. Accordingly, inhibition ofhistone deacetylase can provide an alternate route for treating cancer,hematological disorders, e.g., hemoglobinopathies, genetic relatedmetabolic disorders, e.g., cystic fibrosis and adrenoleukodystrophy,autosomal dominant disorders, e.g. Huntington's disease and spinalmuscular atrophy, and to stimulate hematopoietic cells ex vivo.

In one aspect, a method of inhibiting histone deacetylation activity incells includes contacting the cells with an effective amount of acompound containing an alpha-ketoepoxide group, wherein the compound isnot trapoxin, thereby treating one or more disorders mediated by histonedeacetylase to stimulate hematopoietic cells ex vivo, and determiningwhether the level of acetylated histones in the treated cells is higherthan in untreated cells under the same conditions. In the method, thecompound can be a compound of formula (I), provided that when each of Y¹and Y², independently, is a bond or CH₂, A is unsubstituted phenyl orheterocyclyl, and L is C₄₋₆, L has at least one double bond or at leastone triple bond. In the method, the compound can be1-oxiranyl-8-phenyl-1-octanone,1-oxiranyl-7-phenyl-2,4,6-heptatrien-1-one, or1-oxiranyl-7-phenoxy-2,4,6-heptatrien-1-one.

A compound has the formula (I):

In formula (I), A is a cyclic moiety selected from the group consistingof C₃₋₁₄ cycloalkyl, 3-14 membered heterocycloalkyl, C₄₋₁₄ cycloalkenyl,3-8 membered heterocycloalkenyl, aryl, or heteroaryl. The cyclic moietycan be optionally substituted with alkyl, alkenyl, alkynyl, alkoxy,hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, thio, alkylthio,arylthio, aralkylthio, acylthio, alkylcarbonyloxy, alkyloxycarbonyl,alkylcarbonyl, alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl.Alternatively, A is a saturated branched C₃₋₁₂ hydrocarbon chain or anunsaturated branched C₃₋₁₂ hydrocarbon chain optionally interrupted by—O—, —S—, —N(R^(a))—, —C(O)—, —N(R^(a))—SO₂—, —SO₂—N(R^(a))—,—N(R^(a))—C(O)—O—, —O—C(O)—N(R^(a))—, —N(R^(a))—C(O)—N(R^(b))—,—O—C(O)—, —C(O)—O—, —O—SO₂—, —SO₂—O—, or —O—C(O)—O—. Each of R^(a) andR^(b), independently, can be hydrogen, alkyl, alkenyl, alkynyl, alkoxy,hydroxylalkyl, hydroxyl, or haloalkyl. Each of the saturated and theunsaturated branched hydrocarbon chain can be optionally substitutedwith alkyl, alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo,haloalkyl, amino, thio, alkylthio, arylthio, aralkylthio, acylthio,alkylcarbonyloxy, alkyloxycarbonyl, alkylcarbonyl, alkylsulfonylamino,arninosulfonyl, or alkylsulfonyl.

In formula (I), each of Y¹ and Y², independently, is —CH₂—, —O—, —S—,—N(R^(c))—, —N(R^(c))—C(O)—O—, —N(R^(c))—C(O)—, —C(O)—N(R^(c))—,—O—C(O)—N(R^(c))—, —N(R^(c))—C(O)—N(R^(d))—, —C(O)—, —C(NR^(c))—,—O—C(O)—O—, or a bond. Each of R^(c) and R^(d), independently, can behydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, orhaloalkyl.

In formula (I), L is a straight C₄₋₁₂ hydrocarbon chain optionallycontaining at least one double bond, at least one triple bond, or atleast one double bond and one triple bond. The hydrocarbon chain can beoptionally substituted with C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄alkoxy, hydroxyl, halo, amino, thio, alkylthio, arylthio, aralkylthio,acylthio, nitro, cyano, C_(3-≡)cycloalkyl, 3-5 memberedheterocycloalkyl, monocyclic aryl, 5-6 membered heteroaryl, C₁₋₄alkylcarbonyloxy, C₁₋₄ alkyloxycarbonyl, C₁₋₄ alkylcarbonyl, or formyl.The hydrocarbon chain can be optionally interrupted by —O—, —N(R^(e))—,—N(R^(e))—C(O)—O—, —O—C(O)—N(R^(e))—, —N(R^(e))—C(O)—N(R^(f))—, or—O—C(O)—O—. Each of R^(e) and R^(f), independently, can be hydrogen,alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl.

In formula (I), X¹ is O or S, and each of R^(g), R^(h), and R^(i),independently, is hydrogen or C₁₋₆ alkyl.

In formula (I), when each of Y¹ and Y², independently, is a bond or CH₂,A is unsubstituted phenyl or heterocyclyl, and L is C₄₋₇, L has at leastone double bond or at least one triple bond. In formula (I), when eachof Y¹ and Y² is a bond, A is unsubstituted phenyl, and L is C₄, L is nota diene.

In certain circumstances: each of R^(g), R^(h), and R^(i) can behydrogen; X¹ can be O; each of Y¹ and Y², independently, can be —CH₂—,—O—, —N(R^(c))—, or a bond; L can be a C₄₋₁₂ hydrocarbon chain, a C₅₋₁₂hydrocarbon chain, a C₅₋₁₀ hydrocarbon chain, or a C₆₋₈ hydrocarbonchain; L can be optionally substituted with C₁₋₂ alkyl, C₁₋₂ alkoxy,hydroxyl, —NH₂, —NH(C₁₋₂ alkyl), or —N(C₁₋₂ alkyl)₂; L can contain atleast one double bond, at least one triple bond, or at least one doublebond and one triple bond; L can be an unsaturated hydrocarbon chaincontaining at least one double bond; the double bond can be in transconfiguration; L can be an unsaturated hydrocarbon chain containing atleast two double bonds; or A is a C₅₋₈ cycloalkenyl, 5-8 memberedheteroalkenyl, phenyl, naphthyl, indanyl, or tetrahydronaphthyloptionally substituted with alkyl alkenyl, alkynyl, alkoxy, hydroxyl,hydroxylalkyl, halo, haloalkyl, or amino.

In formula (I), the compound can be1-oxiranyl-7-phenyl-2,4,6-heptatrien-1-one, or1-oxiranyl-7-phenoxy-2,4,6-heptatrien-1-one.

In certain circumstances, A is phenyl, Y¹ is a bond, and L is a C₆₋₁₂hydrocarbon chain containing three double bonds and the carbon adjacentto Y¹ is substituted with phenyl. In other circumstances, A is phenyl,Y¹ is a bond, and L is a C₃₋₁₂ hydrocarbon chain and the carbon adjacentto Y¹ is substituted with two phenyls.

A salt of any of the compounds can be prepared. For example, apharmaceutically acceptable salt can be formed when an amino-containingcompound of this invention reacts with an inorganic or organic acid.Some examples of such an acid include hydrochloric acid, hydrobromicacid, hydroiodic acid, sulfuric acid, phosphoric acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, and acetic acid. Examples of pharmaceutically acceptablesalts thus formed include sulfate, pyrosulfate bisulfate, sulfite,bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate,propionate, decanoate, caprylate, acrylate, formate, isobutyrate,caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, and maleate. A compound of this invention may alsoform a pharmaceutically acceptable salt when a compound of thisinvention having an acid moiety reacts with an inorganic or organicbase. Such salts include those derived from inorganic or organic bases,e.g., alkali metal salts such as sodium, potassium, or lithium salts;alkaline earth metal salts such as calcium or magnesium salts; orammonium salts or salts of organic bases such as morpholine, piperidine,pyridine, dimethylamine, or diethylamine salts.

It should be recognized that a compound of the invention can containchiral carbon atoms. In other words, it may have optical isomers ordiastereoisomers.

Alkyl is a straight or branched hydrocarbon chain containing 1 to 10(preferably, 1 to 6; more preferably 1 to 4) carbon atoms. Examples ofalkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylhexyl, and3-ethyloctyl.

Alkenyl and alkynyl refer to a straight or branched hydrocarbon chaincontaining 2 to 10 carbon atoms and one or more (preferably, 1-4 or morepreferably 1-2) double or triple bonds, respectively. Some examples ofalkenyl and alkynyl are allyl, 2-butenyl, 2-pentenyl, 2-hexenyl,2-butynyl, 2-pentynyl, and 2-hexynyl.

Cycloalkyl is a monocyclic, bicyclic or tricyclic alkyl group containing3 to 14 carbon atoms. Some examples of cycloalkyl are cyclopropyl,cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl.Heterocycloalkyl is a cycloalkyl group containing at least oneheteroatom (e.g., 1-3) such as nitrogen, oxygen, or sulfur. The nitrogenor sulfur may optionally be oxidized and the nitrogen may optionally bequaternized. Examples of heterocycloalkyl include piperidinyl,piperazinyl, tetrahydropyranyl, tetrahydrofuryl, and morpholinyl.Cycloalkenyl is a cycloalkyl group containing at least one (e.g., 1-3)double bond. Examples of such a group include cyclopentenyl,1,4-cyclohexa-di-enyl, cycloheptenyl, and cyclooctenyl groups. By thesame token, heterocycloalkenyl is a cycloalkenyl group containing atleast one heteroatom selected from the group of oxygen, nitrogen orsulfur.

Aryl is an aromatic group containing a 5-14 member ring and can containfused rings, which may be saturated, unsaturated, or aromatic. Examplesof an aryl group include phenyl, naphthyl, biphenyl, phenanthryl, andanthracyl. If the aryl is specified as “monocyclic aryl,” if refers toan aromatic group containing only a single ring, i.e., not a fused ring.

Heteroaryl is aryl containing at least one (e.g., 1-3) heteroatom suchas nitrogen, oxygen, or sulfur and can contain fused rings. Someexamples of heteroaryl are pyridyl, furanyl, pyrrolyl, thienyl,thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, andbenzthiazolyl.

The cyclic moiety can be a fused ring formed from two or more of thejust-mentioned groups. Examples of a cyclic moiety having fused ringsinclude fluorenyl, dihydro-dibenzoazepine. dibenzocycloheptenyl,7H-pyrazino[2,3 -c]carbazole, or 9,10-dihydro-9,10-[2]buteno-anthracene.

Amino protecting groups and hydroxy protecting groups are well-known tothose in the art. In general, the species of protecting group is notcritical, provided that it is stable to the conditions of any subsequentreaction(s) on other positions of the compound and can be removedwithout adversely affecting the remainder of the molecule. In addition,a protecting group may be substituted for another after substantivesynthetic transformations are complete. Examples of an amino protectinggroup include, but not limited to, carbamates such as2,2,2-trichloroethylcarbamate or tertbutylcarbamate. Examples of ahydroxyl protecting group include, but not limited to, ethers such asmethyl, t-butyl, benzyl, p-methoxybenzyl, p-nitrobenzyl, allyl, trityl,methoxymethyl, 2-methoxypropyl, methoxyethoxymethyl, ethoxyethyl,tetrahydropyranyl, tetrahydrothiopyranyl, and trialkylsilyl ethers suchas trimethylsilyl ether, triethylsilyl ether, dimethylarylsilyl ether,triisopropylsilyl ether and t-butyldimethylsilyl ether; esters such asbenzoyl, acetyl, phenylacetyl, formyl, mono-, di-, and trihaloacetylsuch as chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl;and carbonates including but not limited to alkyl carbonates having fromone to six carbon atoms such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl; isobutyl, and n-pentyl; alkyl carbonates having fromone to six carbon atoms and substituted with one or more halogen atomssuch as 2,2,2-trichloroethoxymethyl and 2,2,2-trichloro-ethyl; alkenylcarbonates having from two to six carbon atoms such as vinyl and allyl;cycloalkyl carbonates having from three to six carbon atoms such ascyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and phenyl orbenzyl carbonates optionally substituted on the ring with one or moreC₁₋₆ alkoxy, or nitro. Other protecting groups and reaction conditionscan be found in T. W. Greene, Protective Groups in Organic Synthesis,(3rd, 1999, John Wiley & Sons, New York, N.Y.).

Note that an amino group can be unsubstituted (i.e., —NH₂),mono-substituted (i.e., —NHR), or di-substituted (i.e., —NR₂). It can besubstituted with groups (R) such as alkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, aralkyl, or heteroaralkyl. Halo refers to fluoro,chloro, bromo, or iodo.

Inhibition of a histone deacetylase in a cell is determined by measuringthe level of acetylated histones in the treated cells and measuring thelevel of acetylated histones in untreated cells and comparing thelevels. If the level of histone acetylation in the treated cellsincreases relative to the untreated cells, histone deacetylase has beeninhibited.

Some disorders or physiological conditions may be mediated byhyperactive histone deacetylase activity. A disorder or physiologicalcondition that is mediated by histone deacetylase refers to a disorderor condition wherein histone deacetylase plays a role in triggering theonset thereof. Examples of such disorders or conditions include, but notlimited to, cancer, hemoglobinopathies (e.g., thalassemia or sickle cellanemia), cystic fibrosis, protozoan infection, spinal muscular atrophy,Huntington's disease, alpha-1 anti-trypsin, retrovirus gene vectorreactivation, wound healing, hair growth, peroxisome biogenesisdisorder, and adrenoleukodystrophy.

Other features or advantages will be apparent from the followingdetailed description of several embodiments, and also from the appendedclaims.

DETAILED DESCRIPTION

The compounds of formula (I) and (II) can generally be preparedaccording to the following methods. Specifically, an alpha-ketoepoxidecan be made by dropwise addition of sodium hydroxide to an aldehyde and1,2-epoxy-3-butanone at a controlled pH of 8.5-9, as shown in Scheme A.

Alternatively, as shown in Scheme B, an alpha-ketoepoxide can beprepared by converting a carboxylic acid to the corresponding Weinrebamide using oxalyl chloride followed by N,O-dimethylhydroxylamine.Subsequently, the Weinreb amide is treated with vinyl Grignard. Theresulting vinyl ketone is oxidized with, for example, withm-chloroperbenzoic acid (mCPBA) or an epoxidation catalyst such asJacobsen's catalyst, to gives the desired alpha-ketoepoxide.

An aldehyde or carboxylic acid-containing compound can be prepared byany known methods in the art. For example, a compound having anunsaturated hydrocarbon chain between A and —C(═X¹)—can be preparedaccording scheme C:

where L′ is a saturated or unsaturated hydrocarbon linker between A and—CH═CH—in a compound of the invention, and A and X¹ has the same meaningas defined above. See Coutrot et al., Syn. Comm. 133-134 (1978).Briefly, butyllithium is added to an appropriate amount of anhydroustetrahydrofuran (THF) at a very low temperature (e.g., −65° C.). Asecond solution having diethylphosphonoacetic acid in anhydrous THF isadded dropwise to the stirred butyllithium solution at the same lowtemperature. The resulting solution is stirred at the same temperaturefor an additional 30-45 minutes which is followed by the addition of asolution containing an aromatic acrylaldehyde in anhydrous THF over 1-2hours. The reaction mixture is then warmed to room temperature andstirred overnight. It is then acidified (e.g., with HCl) which allowsthe organic phase to be separated. The organic phase is then dried,concentrated, and purified (e.g., by recrystallization) to form anunsaturated carboxylic acid.

Alternatively, a carboxylic acid-containing compound can be prepared byreacting an acid ester of the formula A-L′—C(═O)—O-lower alkyl with aGrignard reagent (e.g., methyl magnesium iodide) and a phosphorusoxychloride to form a corresponding aldehyde, which can be furtheroxidized (e.g., by reacting with silver nitrate and aqueous NaOH) toform an unsaturated carboxylic acid.

Other types of carboxylic acid-containing compounds (e.g., thosecontaining a linker with multiple double bonds or triple bonds) can beprepared according to published procedures such as those described, forexample, in Parameswara et al., Synthesis, 815-818 (1980) and Denny etal., J. Org. Chem., 27, 3404 (1962). As to compounds wherein X¹ is S,they can be prepared according to procedures described in Sandler, S. R.and Karo, W., Organic Functional Group Preparations, Volume III(Academic Press, 1972) at pages 436-437. Additional synthetic methodscan be found in March, J. Advanced Organic Chemistry, 4^(th) ed., (WileyInterscience, 1992).

Note that appropriate protecting groups may be needed to avoid formingside products during the preparation of a compound of the invention. Forexample, if the linker L′ contains an amino substituent, it can be firstprotected by a suitable amino protecting group such as trifluoroacetylor tert-butoxycarbonyl prior to being treated with reagents such asbutyllithium. See, e.g., T. W. Greene, supra, for other suitableprotecting groups.

A compound produced by the methods shown above can be purified by flashcolumn chromatography, preparative high performance liquidchromatography, or crystallization.

A pharmaceutical composition including the compound described above canbe used to inhibit histone deacetylase in cells and can be used to treatdisorders associated with abnormal histone deacetylase activity. Someexamples of these disorders are cancers (e.g., leukemia, lung cancer,colon cancer, CNS cancer, melanoma, ovarian cancer, cervical cancer,renal cancer, prostate cancer, and breast cancer), hematologicaldisorders (e.g., hemoglobinopathies, thalassemia, and sickle cellanemia) and genetic related metabolic disorders (e.g., cystic fibrosis,spinal muscular atrophy, peroxisome biogenesis disorder, alpha-1anti-trypsin, and adrenoleukodystrophy). The compounds described abovecan also stimulate hematopoietic cells ex vivo, ameliorating protozoalparasitic infection, accelerate wound healing, and protecting hairfollicles.

An effective amount is defined as the amount which is required to confera therapeutic effect on the treated patient, and is typically determinedbased on age, surface area, weight, and condition of the patient. Theinterrelationship of dosages for animals and humans (based on milligramsper meter squared of body surface) is described by Freireich et al.,Cancer Chemother. Rep. 50, 219 (1966). Body surface area may beapproximately determined from height and weight of the patient. See,e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537(1970). An effective amount of a compound described herein can rangefrom about 1 mg/kg to about 300 mg/kg. Effective doses will also vary,as recognized by those skilled in the art, dependent on route ofadministration, excipient usage, and the possibility of co-usage,pre-treatment, or post-treatment, with other therapeutic treatmentsincluding use of other chemotherapeutic agents and radiation therapy.Other chemotherapeutic agents that can be co-administered (eithersimultaneously or sequentially) include, but not limited to, paclitaxeland its derivatives (e.g., taxotere), doxorubicin, L-asparaginase,dacarbazine, amascrine, procarbazine, hexamethylmelamine, mitoxantrone,and gemicitabine.

The pharmaceutical composition may be administered via the parenteralroute, including orally, topically, subcutaneously, intraperitoneally,intramuscularly, and intravenously. Examples of parenteral dosage formsinclude aqueous solutions of the active agent, in a isotonic saline, 5%glucose or other well-known pharmaceutically acceptable excipient.Solubilizing agents such as cyclodextrins, or other solubilizing agentswell-known to those familiar with the art, can be utilized aspharmaceutical excipients for delivery of the therapeutic compounds.Because some of the compounds described herein can have limited watersolubility, a solubilizing agent can be included in the composition toimprove the solubility of the compound. For example, the compounds canbe solubilized in polyethoxylated castor oil (Cremophor EL®) and mayfurther contain other solvents, e.g., ethanol. Furthermore, compoundsdescribed herein can also be entrapped in liposomes that may containtumor-directing agents (e.g., monoclonal antibodies having affinitytowards tumor cells).

A compound described herein can be formulated into dosage forms forother routes of administration utilizing conventional methods. Forexample, it can be formulated in a capsule, a gel seal, or a tablet fororal administration. Capsules may contain any standard pharmaceuticallyacceptable materials such as gelatin or cellulose. Tablets may beformulated in accordance with conventional procedures by compressingmixtures of a compound described herein with a solid carrier and alubricant. Examples of solid carriers include starch and sugarbentonite. Compounds of this invention can also be administered in aform of a hard shell tablet or a capsule containing a binder, e.g.,lactose or mannitol, a conventional filler, and a tableting agent.

The activities of a compound described herein can be evaluated bymethods known in the art, e.g., MTT(3-[4,5-dimehtythiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay,clonogenic assay, ATP assay, or Extreme Drug Resistance (EDR) assay. SeeFreuhauf, J. P. and Manetta, A., Chemosensitivity Testing in GynecologicMalignancies and Breast Cancer 19, 39-52 (1994). The EDR assay, inparticular, is useful for evaluating the antitumor and antiproliferativeactivity of a compound described herein. Cells are treated for four dayswith a compound. Both untreated and treated cells are pulsed withtritiated thymidine for 24 hours. Radioactivity of each type of cells isthen measured and compared. The results are then plotted to generatedrug response curves, which allow IC₅₀ values (the concentration of acompound required to inhibit 50% of the population of the treated cells)to be determined.

Histone deacetylase inhibitory activity can be measured based onprocedures described by Hoffmann et al., Nucleic Acids Res., 27,2057-2058 (1999). Briefly, the assay starts with incubating the isolatedhistone deacetylase enzyme with a compound of the invention, followed bythe addition of a fluorescent-labeled lysine substrate (contains anamino group at the side chain which is available for acetylation). HPLCis used to monitor the labeled substrate. The range of activity of eachtest compound is preliminarily determined using results obtained fromHPLC analyses. IC₅₀ values can then be determined from HPLC resultsusing different concentrations of compounds of this invention. Allassays are duplicated or triplicated for accuracy. The histonedeacetylase inhibitory activity can be compared with the increasedactivity of acetylated histone for confirmation.

Compounds of this invention are also evaluated for effects on treatingX-linked adrenoleukodystrophy (X-ALD), a peroxisomal disorder withimpaired very long-chain fatty acid (VLCFA) metabolism. In such anassay, cell lines derived from human primary fibroblasts and(EBV-transformed lymphocytes) derived from X-ALD patients grown on RPMIare employed. Tissue culture cells are grown in the presence or absenceof test compounds. For VLCFA measurements, total lipids are extracted,converted to methyl esters, purified by TLC and subjected to capillaryGC analysis as described in Moser et al., Technique in DiagnosticBiochemical Genetics: A Laboratory Manual (ed. A., H. F.) 177-191(Wiley-Liss, New York, 1991). C24:0 β-oxidation activity oflymphoclastoid cells are determined by measuring their capacity todegrade [1-¹⁴C]-C24:0 fatty acid to water-soluble products as describedin Watkins et al., Arch. Biochem. Biophys. 289, 329-336 (1991). Thestatistical significance of measured biochemical differences betweenuntreated and treated X-ALD cells can be determined by a two-tailedStudent's t-test.

Further, compounds of the present invention are evaluated for theireffects in treating cystic fibrosis (CF). Since the initial defect inthe majority of cases of CF is the inability of mutant CF protein (CFTR)to fold properly and exit the ER, compounds of the invention are testedto evaluate their efficacy in increasing the trafficking of the CFprotein out of the ER and its maturation through the Golgi. During itsbiosynthesis, CFTR is initially synthesized as a nascent polypeptidechain in the rough ER, with a molecular weight of around 120 kDa (BandA). It rapidly receives a core glycosylation in the ER, giving it amolecular weight of around 140 kDa (Band B). As CFTR exits the ER andmatures through the Golgi stacks, its glycosylation is modified until itachieves a terminal mature glycosylation, affording it a molecularweight of around 170 kDa (Band C). Thus, the extent to which CFTR exitsthe ER and traverses the Golgi to reach the plasma membrane may bereflected in the ratio of Band B to Band C protein. CFTR isimmunoprecipitated from control cells, and cells exposed to testcompounds. Both wt CFTR and ΔF508 CFTR expressing cells are tested.Following lysis, CFTR are immunoprecipitated using various CFTRantibodies. Immunoprecipitates are then subjected to in vitrophosphorylation using radioactive ATP and exogenous protein kinase A.Samples are subsequently solubilized and resolved by SDS-PAGE. Gels arethen dried and subject to autoradiography and phosphor image analysisfor quantitation of Bands B and C are determined on a BioRad personalfix image station.

Furthermore, compounds of this invention can be used to treat homozygousβ thalassemia, a disease in which there is inadequate production of βglobin leading to severe anemia. See Collins et al., Blood, 85(1), 43-49(1995).

Still further, compounds of the present invention are evaluated fortheir use as antiprotozoal or antiparasitic agents. The evaluation canbe conducted using parasite cultures (e.g., Asexual P. falciparum). SeeTrager, W. & Jensen, J. B., Science 193, 673-675 (1976). Test compoundsare dissolved in dimethyl sulfoxide (DMSO) and added to wells of aflat-bottomed 96-well microtitre plate containing human serum. Parasitecultures are then added to the wells, whereas control wells only containparasite cultures and no test compound. After at least one invasioncycle, and addition of labeled hypoxanthine monohydrochloride, the levelof incorporation of labeled hypoxanthine is detected. IC₅₀ values can becalculated from data using a non-linear regression analysis.

The toxicity of a compound described herein is evaluated when a compoundof the invention is administered by single intraperitoneal dose to testmice. After administration of a predetermined dose to three groups oftest mice and untreated controls, mortality/morbidity checks are madedaily. Body weight and gross necropsy findings are also monitored. Forreference, see Gad, S. C. (ed.), Safety Assessment for Pharmaceuticals(Van Nostrand Reinhold, New York, 1995).

The following specific examples, which described syntheses, screening,and biological testing of various compounds of this invention, aretherefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever. All publicationsrecited herein, including patents, are hereby incorporated by referencein their entirety.

EXAMPLES Synthesis of 5-Phenyl-2,4-Pentadienal

To a cooled (0-5° C.) 927 mL of 1 M solution of phenyl magnesium bromidein tetrahydrofuran was added dropwise a solution of crotonaldehyde (65.0g) in 130 mL of anhydrous ether over a period of 2 hours and 45 minutes.The reaction was stirred for an additional 45 minutes and then warmed toroom temperature. After four more hours of stirring, saturated ammoniumchloride aqueous solution (750 mL) was added to the reaction. Themixture was extracted with 750 mL of ether twice. The combined extractwas dried over anhydrous potassium carbonate and filtered. The solventwas evaporated to give 135.88 g (99.9%) of the desired1-phenyl-2-buten-1-ol as an oil which was used in the next step withoutfurther purification.

1-Phenyl-2-buten-1-ol (135.88 g) was dissolved in 2300 mL of dioxane andtreated with 2750 mL of dilute hydrochloric acid (2.3 mL of concentratedhydrochloric acid in 2750 mL of water) at room temperature. The mixturewas stirred overnight and then poured into 4333 mL of ether andneutralized with 2265 mL of saturated aqueous sodium bicarbonate. Theaqueous phase was extracted with 1970 mL of ether. The combined extractwas dried over anhydrous potassium carbonate. Evaporation of the solventfollowed by Kugelrohr distillation at 30° C. for 30 minutes afforded131.73 g (96.8%) of the desired 4-phenyl-3-buten-2-ol as an oil whichwas used in the next step without further purification.

Dimethylformamide (DMF, anhydrous, 14 mL) was cooled to 0-5° C. andphosphorus oxychloride (8.2 mL) was added dropwise over a period of 40minutes. The resulting solution was added dropwise to a cooled (0-5° C.)solution of 4-phenyl-3-buten-2-ol (10 g) in 32 mL of anhydrous DMF overa period of an hour. The reaction mixture was warmed to room temperatureover a 35-minute period and then gradually heated up to 80° C. over aperiod of 45 minutes. The reaction was stirred at 80° C. for three hoursand then cooled to 0-5° C. To the cooled reaction solution was addeddropwise a solution of sodium acetate (40 g) in deionized water (100 mL)over a period of one hour. The mixture was then reheated to 80° C.,stirred at 80° C. for an additional 10 minutes, cooled down to roomtemperature and extracted with ether (100 mL) twice. The combinedextract was washed with brine (100 mL), dried over anhydrous sodiumsulfate, filtered and concentrated under vacuum to yield 8.78 g of thedesired 5-phenyl-2,4-pentadienal as a liquid which was used in the nextstep without further purification. ¹H NMR (CDCl₃, 300 MHz), δ(ppm) 7.51(m, 2H), 7.37 (m, 3H), 7.26 (m, 1H), 7.01 (m, 2H), 6.26 (m, 1H). Thesynthesis is summarized in Scheme I.

Synthesis of 5-Phenoxy-2,4-Pentadienal

2-Formylvinyl phenyl ether is prepared by treating phenoxyacetaldehydewith formaldehyde and diethylamine hydrochloride salt. The ether is thenreacting with a solution of diethylphosphonoacetic acid andn-butyllithium in anhydrous tetrahydrofuran (THF) to form5-phenoxy-2,4-pentadienoic acid. 5-Phenyl-2,4-pentadienal is obtained byfirst converting the carboxylic acid to a Weinreb amide using oxalylchloride followed by N,O-dimethylhydroxylamine. Subsequently, reductionof the Weinreb amide with lithium aluminum hydride (LAH) in THF leads tothe formation of 5-phenoxy-2,4-pentadienal. The synthesis is summarizedin Scheme II.

Synthesis of 1,2-Epoxy-3-Butanone

1,2-Epoxy-3-butanone was prepared by treating methyl vinyl ketone inmethanol with 30% of hydrogen peroxide followed by 10% aqueous sodiumhydroxide, as shown below.

Synthesis of 1-Oxiranyl-7-Phenyl-2,4,6-Heptatrien-1-One

1-Oxiranyl-7-phenyl-2,4,6-heptatrien-1-one is made by dropwise additionof 5% aqueous sodium hydroxide to a mixture of 5-phenyl-2,4-pentadienaland 1,2-epoxy-3-butanone at a controlled pH of 8.5-9, as shown in SchemeIIIA.

Alternatively, as shown in Scheme IV,1-oxiranyl-7-phenyl-2,4,6-heptatrien-1-one can be prepared by converting7-phenyl-2,4,6-heptatrienoic acid to the corresponding Weinreb amideusing oxalyl chloride followed by N,O-dimethylhydroxylamine.Subsequently, oxidation of the Weinreb amide with m-chloroperbenzoicacid (mCPBA) gives the desired alpha-keto epoxide.

Synthesis of 1-Oxiranyl-7-Phenoxy-2,4,6-Heptatrien-1-One

1-Oxiranyl-7-phenoxy-2,4,6-heptatrien-1-one is made by dropwise additionof 5% aqueous sodium hydroxide to a mixture of 5-phenoxy-2,4-pentadienaland 1,2-epoxy-3-butanone at a controlled pH of 8.5-9, as shown in SchemeIIIB.

Assays

Compounds selected from 1-oxiranyl-8-phenyl-1-octanone (preparedaccording to the procedure described in Bioorg. & Med. Chem. Lett., 9(1999), 2283-2288), 1-oxiranyl-7-phenyl-2,4,6-heptatrien-1-one, or1-oxiranyl-7-phenoxy-2,4,6-heptatrien-1-one are used in the assaysdescribed below.

In vitro Efficacy Studies—Extreme Drug Resistance (EDR) Assay

The PC3 cell line is maintained in RPMI supplemented with 10% fetal calfserum and antibiotics. Cells are suspended in 0.12% soft agar incomplete medium and plated (2,000 cells per well) in different drugconcentrations onto a 0.4% agarose underlayer in 24-well plates. Platingcalls on agarose underlayers supports the proliferation only of thetransformed cells, ensuring that the growth signal stems from themalignant component of the tumor.

All compounds are dissolved in DMSO to 200× stock solutions. Stocksolutions are diluted to 20× working solutions using the tissue culturemedium, then are serially diluted and added to the 24-well plates. Theinitial range of concentrations is 1 micromolar to 200 micromolar. Nosignificant changes in pH of the culture medium are observed under theabove conditions. Diluent control wells contain PC3 cells treated withDMSO, at the dilutions used for appropriate drug treatment. Allexperimental points are represented by two separate wells (duplicates).Four wells containing tumor cells that are not treated with drugs serveas negative controls in each experiment.

Cells are incubated with drugs under standard culture conditions for 5days. Cultures are pulsed with tritiated thymidine (³H-TdR, New LifeScience Products, Boston, Mass.) at 5 μCi per well for the last 48 hoursof the culture period. Cell culture plates are then heated to 90° C. toliquefy the agarose, and cells are harvested onto glass fiber filters,which are then placed into counting vials containing liquidscintillation fluid. The radioactivity trapped on the filters is countedwith a Beckman scintillation counter. The fraction of surviving cells isdetermined by comparing ³H-TdR incorporation in treated (experimentalpoints) and untreated (negative control) wells. Microsoft Excel is usedto organize the raw data on EDR experiments, and the SigmaPlot programis utilized to generate drug response curves. All drug response curvesare approximated as sigmoidal equations (characteristic for typical drugresponse curves) to fit the data. IC₅₀ values are determined using theapproximated sigmoidal curves and expressed as μM.

Histone (Hyper)Acetylation Assay

The effect of a compound described herein on histone acetylation can beevaluated in an assay using mouse erythroleukemia cells. Studies areperformed with the DS19 mouse erythroleukemia cells maintained in RPMI1640 medium with 25 mM HEPES buffer and 5% fetal calf serum. The cellsare incubated at 37° C.

Histones are isolated from cells after incubation for periods of 2 and24 hours. The cells are centrifuged for 5 minutes at 2000 rpm in theSorvall SS34 rotor and washed once with phosphate buffered saline. Thepellets are suspended in 10 mL lysis buffer (10 mM Tris, 50 mM sodiumbisulfite, 1% Triton X-100, 10 mM magnesium chloride, 8.6% sucrose, pH6.5) and homogenized with six strokes of a Teflon pestle. The solutionis centrifuged and the pellet washed once with 5 mL of the lysis bufferand once with 5 mL 10 mM Tris, 13 mM EDTA, pH 7.4. The pellets areextracted with 2×1 mL 0.25 N HCl. Histones are precipitated from thecombined extracts by the addition of 20 mL acetone and refrigerationovernight. The histones are pelleted by centrifuging at 5000 rpm for 20minutes in the Sorvall SS34 rotor. The pellets are washed once with 5 mLacetone and protein concentration are quantitated by the Bradfordprocedure.

Separation of acetylated histones is usually performed with an aceticacid-urea polyacrylamide gel electrophoresis procedure. Resolution ofacetylated H4 histones is achieved with 6.25 N urea and no detergent asoriginally described by Panyim and Chalkley, Arch. Biochem. Biophys.130, 337-346 (1969). 25 μg Total histones are applied to a slab gelwhich is run at 20 mA. The run is continued for a further two hoursafter the Pyronin Y tracking dye has run off the gel. The gel is stainedwith Coomassie Blue R. The most rapidly migrating protein band is theunacetylated H4 histone followed by bands with 1, 2, 3 and 4 acetylgroups which can be quantitated by densitometry. The procedure fordensitometry involves digital recording using the Alpha Imager 2000,enlargement of the image using the PHOTOSHOP program (Adobe Corp.) on aMACINTOSH computer (Apple Corp.), creation of a hard copy using a laserprinter and densitometry by reflectance using the Shimadzu CS9000Udensitometer. The percentage of H4 histone in the various acetylatedstates is expressed as a percentage of the total H4 histone.

The concentration of a compound of the invention required to decreasethe unacetylated H4 histone by 50% (i.e., EC₅₀) can then be determinedfrom data obtained using different concentrations of test compounds.

Histone Deacetylation Assay

The determination of the inhibition of histone deacetylase by compoundsdescribed herein is based upon the procedure described by Hoffmann etal., Nucleic Acids Res. 27, 2057-2058 (1999). The histone deacetylase isisolated from rat liver as previously described in Kolle, D. et al.Methods: A Companion to Methods in Enzymology 15: 323-331 (1998).Compounds are initially dissolved in either ethanol or in DMSO toprovide a working stock solution. The synthetic substrate used in theassay isN-(4-methyl-7-coumarinyl)-N-α(tert-butyloxy-carbonyl)-N-Ω-acetyllysineamide(MAL).

The assay is performed in a final total volume of 120 μL consisting of100 μL of 15 mM tris-HCl buffer at pH 7.9 and 0.25 mM EDTA, 10 mM NaCl,10% glycerol, 10 mM mercaptoethanol and the enzyme. The assay isinitiated upon the addition of 10 μL of a test compound followed by theaddition of a fluorescent-labeled lysine substrate to each assay tube inan ice bath for 15 minutes. The tubes are transferred to a water bath at37° C. for an additional 90 minutes.

An initial assay is performed to determine the range of activity of eachtest compound. The determination of IC₅₀ values is made from the resultsof five dilutions in range according to the expected potency for eachtest compound. Each assay is duplicated or triplicated.

Other embodiments are within the scope of the following claims.

1. A method of inhibiting histone deacetylation activity in cellscomprising contacting the cells with an effective amount of a compoundcontaining an alpha-ketoepoxide group, wherein the compound is nottrapoxin, thereby treating one or more disorders mediated by histonedeacetylase or stimulating hematopoietic cells ex vivo, and determiningwhether the level of acetylated histones in the treated cells is higherthan in untreated cells under the same conditions.
 2. The method ofclaim 1, wherein the compound is of formula (I):

wherein A is a cyclic moiety selected from the group consisting of C₃₋₁₄cycloalkyl, 3-14 membered heterocycloalkyl, C₄₋₁₄ cycloalkenyl, 3-8membered heterocycloalkenyl, aryl, and heteroaryl; the cyclic moietybeing optionally substituted with alkyl, alkenyl, alkynyl, alkoxy,hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, thio, alkylthio,arylthio, aralkylthio, acylthio, alkylcarbonyloxy, alkyloxycarbonyl,alkylcarbonyl, alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl; or Ais a saturated branched C₃₋₁₂ hydrocarbon chain or an unsaturatedbranched C₃₋₁₂ hydrocarbon chain optionally interrupted by —O—, —S—,—N(R^(a))—, —C(O)—, —N(R^(a))—SO₂—, —SO₂—N(R^(a))—, —N(R^(a))—C(O)—O—,—O—C(O)—N(R^(a))—, —N(R^(a))—C(O)—N(R^(b))—, —O—C(O)—, —C(O)—O—,—O—SO₂—, —SO₂—O—, or —O—C(O)—O—, where each of R^(a) and R^(b),independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy,hydroxylalkyl, hydroxyl, or haloalkyl; each of the saturated and theunsaturated branched hydrocarbon chain being optionally substituted withalkyl, alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo,haloalkyl, amino, thio, alkylthio, arylthio, aralkylthio, acylthio,alkylcarbonyloxy, alkyloxycarbonyl, alkylcarbonyl, alkylsulfonylamino,aminosulfonyl, or alkylsulfonyl; each of Y¹ and Y², independently, is—CH₂—, —O—, —S—, —N(R^(c))—, —N(R^(c))—C(O)—O—, —N(R^(c))—C(O)—,—C(O)—N(R^(c))—, —O—C(O)—N(R^(c))—, —N(R^(c))—C(O)—N(R^(d))—, —C(O)—,—C(NR^(c))—, —O—C(O)—O—, or a bond; each of R^(c) and R^(d),independently, being hydrogen, alkyl, alkenyl, alkynyl, alkoxy,hydroxylalkyl, hydroxyl, or haloalkyl; L is a straight C₃₋₁₂ hydrocarbonchain optionally containing at least one double bond, at least onetriple bond, or at least one double bond and one triple bond; thehydrocarbon chain being optionally substituted with C₁₋₄ alkyl, C₂₋₄alkenyl, C₂₄ alkynyl, C₁₋₄ alkoxy, hydroxyl, halo, amino, thio,alkylthio, arylthio, aralkylthio, acylthio, nitro, cyano, C₃₋₅cycloalkyl, 3-5 membered heterocycloalkyl, monocyclic aryl, 5-6 memberedheteroaryl, C₁₋₄ alkylcarbonyloxy, C₁₋₄ alkyloxycarbonyl, C₁₋₄alkylcarbonyl, or formyl; and further being optionally interrupted by—O—, —N(R^(e))—, —N(R^(e))—C(O)—O—, —O—C(O)—N(R^(e))—,—N(R^(e))—C(O)—N(R^(f))—, or —O—C(O)—O—; each of R^(e) and R^(f,)independently, being hydrogen, alkyl, alkenyl, alkynyl, alkoxy,hydroxylalkyl, hydroxyl, or haloalkyl; X¹ is O or S; and each of R^(g),R^(h), and R^(i), independently, is hydrogen or C₁₋₆ alkyl; providedthat when each of Y¹ and Y², independently, is a bond or CH₂, A isunsubstituted phenyl or heterocyclyl, and L is C₄₋₆, L has at least onedouble bond or at least one triple bond; or a salt thereof.
 3. Themethod of claim 1, wherein the disorder is selected from the groupconsisting of cancer, hemoglobinopathies, thalassemia, sickle cellanemia, cystic fibrosis, protozoan infection, spinal muscular atrophy,Huntington's disease, alpha-1 anti-trypsin, retrovirus gene vectorreactivation, wound healing, hair growth, peroxisome biogenesisdisorder, and adrenoleukodystrophy.
 4. The method of claim 2, whereinthe disorder is selected from the group consisting of cancer,hemoglobinopathies, thalassemia, sickle cell anemia, cystic fibrosis,protozoan infection, spinal muscular atrophy, Huntington's disease,alpha-1 anti-trypsin, retrovirus gene vector reactivation, woundhealing, hair growth, peroxisome biogenesis disorder, andadrenoleukodystrophy.
 5. The method of claim 1, wherein the disorder iscancer, cystic fibrosis, or adrenoleukodystrophy.
 6. The method of claim2, wherein the disorder is cancer, cystic fibrosis, oradrenoleukodystrophy.
 7. The method of claim 1, wherein hematopoieticcells are stimulated ex vivo.
 8. The method of claim 2, whereinhematopoietic cells are stimulated ex vivo.
 9. The method of claim 1,wherein the compound is 1-oxiranyl-8-phenyl-1-octanone,1-oxiranyl-7-phenyl-2,4,6-heptatrien-1-one, or1-oxiranyl-7-phenoxy-2,4,6-heptatrien-1-one.